Bioluminescent bacteria are widely found in both marine and terrestrial environments. Interestingly, all identified species of naturally occurring marine and terrestrial bioluminescent bacteria are Gram-negative. To date, at least eleven species in four Gram-negative genera have been described: Vibrio, Photobacterium, Shewanella (Altermonas) and Photorhabdus (Xenorhabdus). In all these species, the five genes responsible for bioluminescence are clustered in the lux operon (luxCDABE).
The bioluminescence (emitted blue-green light having a wavelength of about 490 nm) is thought to result from a luciferase-catalyzed oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain fatty aldehyde. The luciferase enzyme is encoded by two subunits (luxAB), whereas the fatty acid reductase polypeptides responsible for the biosynthesis of the aldehyde substrate for the luminescent reaction are encoded by the three genes luxCDE. The genes encoding luciferase and the fatty acid reductase polypeptides have been cloned from the lux operons of Vibrio, Photobacterium and Photorhabdus and sequenced. In each case, the luxCDE genes flank the luxAB genes, with transcription in the order luxCDABE. Although a number of additional lux genes have been identified in each of these three bacteria, only luxA-E are essential for the biosynthesis of light (reviewed by Meighen, E., (1993, The FASEB Journal 7:1016–1022 and Ulitzur, S., (1997), J. Biolumin Chemilumin 12:179–192).
Methods described in U.S. Pat. No. 5,650,135, make possible the detection of bioluminescent bacteria in a living animal without dissecting or otherwise opening the animal up (“in vivo monitoring”)—the light is detected through muscle, skin, fur & other traditionally “opaque” tissues using a highly sensitive camera
While a non-bioluminescent Gram-negative bacterium can typically be engineered to have bioluminescence properties by cloning into it a luxCDABE operon (under control of a suitable promoter) from a bioluminescent species (see, e.g., Contag, et al., U.S. Pat. No. 5,650,135), previous attempts to make bioluminescent Gram positive bacteria have met with limited success. For example, one approach employed an expression cassette encoding a functional LuxAB fusion protein (Jacobs, M., et al., (1991) Mol. Gen. Genet. 230:251–256). In this cassette, a Gram-positive ribosome binding site (RBS) was inserted upstream of luxA, with the luxB gene cloned in frame downstream of luxA. Although this approach has been successful in generating a number of novel genera of bioluminescent Gram-positive bacteria useful for certain environmental and food safety studies (e.g., the assessment of food products for contamination by such bacteria), these bacteria are not useful for studying pathogenicity. A major reason for this limitation is that the LuxAB fusion proteins described in the prior art are not stable at mammalian body temperatures, and are thus capable of catalyzing only minimal light production in bacterial cells at 37° C.
In fact, none of the bioluminescent Gram-positive bacteria which have been published to date produce enough light in vivo to make them useful for the in-vivo monitoring applications discussed above.
The present invention provides, inter alia, such methods, transposon cassettes, and other tools useful for generating bioluminescent bacteria, for example, Gram-positive bacteria and related organisms, suitable for studies relating to infection and/or pathogenesis.